The invention relates to the field of electroporation and transformation of host cells, particularly bacterial cells.
Introducing nucleic acids into E. coli and other host organisms is central to many types of experiments and analyses. For example, when searching for a gene of interest in a DNA library, the library must be transferred into a host organism. Since the DNA of many organisms is very complex, the number of independent clones that are needed to completely represent the organism is large. In order to create a library that completely represents the organism, the efficiency at which the DNA can be introduced into the host cell becomes limiting. By optimizing this process, the ability to create and screen DNA libraries is facilitated.
Similarly, many other experimental analyses are limited by the ability to introduce DNA into a host organism. When cloning large segments of DNA for whole genome analysis (i.e., using bacterial artificial chromosomes), when performing PCR cloning, or when carrying out random mutagenesis of a gene, followed by cloning all potential altered forms, success often depends on the size of the initial transformation pool. Again, developing conditions that improve the process of introducing nucleic acids into a host organism increases the chance that the experiment will succeed.
There are several methods for introducing nucleic acids into various host cells, e.g., incubating the host cells with co-precipitates of nucleic acids (Graham and van der Eb, Virology, 52: 456-467 (1973)), directly injecting genes into the nucleus of the host cells (Diacumakos, Methods in Cell Biology, Vol. 7, eds. Prescott, D. M. (Academic Press) pp. 287-311 (1973), introducing nucleic acids via viral vectors (Hamer and Leder, Cell, 18: 1299-1302 (1979)), and using liposomes as a means of gene transfer (Fraley et al., J. Biol. Chem., 255: 10431-10435 (1980); Wong et al. Gene, 10: 87-94 (1980)). Electroporation has also been used to transform host organisms, including E. coli. (Dower et al., Nucleic Acids Research, 16: 6127-6145 (1988); Taketo, Biochimica et Biophysica Acta, 949: 318-324 (1988); Chassy and Flickinger, FEMS Microbiology Letters, 44: 173-177 (1987); and Harlander, Streptococcal Genetics, eds. Ferretti and Curtiss (American Society of Microbiology, Washington, D.C.) pp. 229-233 (1987)).
In general, electroporation involves the transfer of genes or gene fragments (nucleic acids) into a host cell by exposure of the cell to a high voltage electric impulse in the presence of the genes or gene fragments (Andreason and Evans, Biotechniques, 6: 650-660 (1988)). Quite often, the genes and gene fragments are exogenous, i.e., heterologous to the host organism. Also, frequently the cells have been stored prior to electroporation. A typical method of storage is to freeze the cells. The cells are frozen at a temperature that preserves viability. After thawing those cells, genes or gene fragments may be transferred by electroporation into the cells, permanently or transiently for short-term expression.
An example of a typical electroporation method is to grow bacteria in enriched media (of any sort) and to concentrate the bacteria by washing in a buffer that contains 10% glycerol (Dower et al., 1988, U.S. Pat. No. 5,186,800). As discussed in U.S. Pat. No. 5,186,800, which is hereby incorporated by reference in its entirety, DNA is added to the cells and the cells are subjected to an electrical discharge, which temporarily disrupts the outer cell wall of the bacterial cells to allow DNA to enter the cells.
The electrical treatment to which the host cells are subjected during the process of electroporation is very harsh and typically results in the death of  greater than 90% of the host cells. However, it is believed that the majority of cells that survive electroporation take up the nucleic acids of interest. The efficiency with which nucleic acid transfer occurs depends on a variety of factors, including the genetic background of the host cells. Routinely, an efficiency of 109xe2x88x921xc3x971010 transformants per xcexcg of input DNA (plasmid pUC18) may be achieved. Using Rec A- cells, typically 5.0xe2x88x927.0xc3x97109 cells are transformed per xcexcg of input DNA. When the host cells are E. coli, 10.% or less of the treated bacteria survive. However, the percentage is significantly lower for certain strains of E. coli that are inefficient at electrotransformation.
In developing and refining electroporation methodology, researchers have identified factors that impact the efficiency of the transfer. These factors include, e.g., the electrical field strength, the pulse decay time, the pulse shape, the temperature in which the electroporation is conducted, the type of cell, the type of suspension buffer, and the concentration and size of the nucleic acid to be transferred (Andreason and Evans, Analytical Biochemistry, 180: 269-275 (1988); Sambrook, et al., Molecular Cloning: a Laboratory Manual, 2nd Edition, eds. Sambrook, et al. (Cold Spring Harbor Laboratory Press) pp. 1.75 and 16.54-16.55 (1989); Dower et al., (1988); Taketo (1988)). Thus, previous attempts to improve the electroporation efficiency have focused on these factors and thus, have primarily involved manipulation of methods used to prepare the cells, e.g., washing and centrifugation of cells during the processing stage, and methods for applying the electrical shock (i.e., different configuration of the apparatus that delivers the electrical pulse).
Typically, researchers have only modified the host cell suspension materials to aid in freezing the cells before the electrical treatment (Taketo 1988).
This invention provides improved methods of electroporation and other electrical treatment of cells. The methods comprise the addition of sugars or sugar derivatives, e.g., sugar alcohols, to host cells suspended in a substantially non-ionic solution, either prior to initial freezing, or after thawing, but prior to electrotransformation. The methods of this invention improve electroporation efficiency. The level of improvement is 30% (for cells that generally exhibit higher efficiency) to 300% (for cells that have lower efficiency).
In certain embodiments, at least one sugar or sugar derivative is added to the host cells suspended in a substantially non-ionic solution prior to electrically treating the host cells. Preferably, the sugar or derivative thereof is added in a concentration range of about 0.1% to about 5%. In certain embodiments of the invention, the host cells are suspended in the non-ionic sugar or sugar derivative solution before they are electrically transformed. In certain embodiments, one may prefer to freeze the cells prior to the electrotransformation. For instance, one may suspend the host cells in the sugar or sugar derivative solution before freezing the cells. In another instance, the cells may be suspended in the sugar or sugar derivative solution after the cells have been frozen and thawed. In certain embodiments, the host cells are bacterial cells, preferably gram-negative bacterial cells, and most preferably, E. coli. 
This invention also provides a kit for use in the practice of the above-described methods of transferring nucleic acids of interest into host cells. The electroporation kit includes host cells suspended in a substantially non-ionic solution comprising at least one sugar or sugar derivative. In certain embodiments, the kit includes host cells suspended in a non-ionic solution having a sugar or sugar derivative a concentration of about 0.1% to about 5%. In other embodiments, the kit includes a non-ionic solution comprising a mixture of sugars and sugar derivatives.
Thus, this invention provides methods for transferring nucleic acids of interest into host cells, comprising the steps of mixing host cells suspended in a substantially non-ionic solution comprising at least one sugar or sugar derivative with nucleic acids of interest, and subjecting the mixture to electrical treatment, thereby permitting the transfer of the nucleic acids of interest into the host cells.
In certain embodiments of the invention, the non-ionic solution includes glycerol or dimethyl sulfoxide.
As used herein, the term xe2x80x9cnon-ionic solutionxe2x80x9d refers to a buffer solution that would have minimal or no ions present. In many instances, non-ionic solutions are also non-polar, therefore, for the purposes of defining terms in this application, solutions that are non-polar are also, non-ionic. The concentration of ions in the buffer is adequately low so that when electricity is discharged into the host cells, little or no additional current is carried into the cells. The presence of ions in the buffer may result in additional current being carried into the cell and can lower the survival rate of the host cells.
A number of sugars and sugar derivatives are known to those skilled in the art. The sugars or sugar derivatives useful in the processes and kits of this invention may be in either the D-stereoisomeric or the L-forms (enantiomers) form. Sugars that may be used in the methods and kits of this invention include, but are not limited to: aldoses, such as monosaccharides which include trioses (i.e. glyceraldehyde), tetroses (i.e. erythrose, threose), pentoses (i.e. arabinose, xylose, ribose, lyxose), hexoses (i.e. glucose, mannose, galactose, idose, gulose, altrose, allose, talose), heptoses (i.e. sedoheptulose), octoses (i.e. glycero-D-manno-octulose), pentose ring sugars (i.e. ribofuranose, ribopyranose); disaccharides (i.e., sucrose, lactose, trehalose, maltose, cellobiose, gentiobiose); and trisaccharides (i.e., raffinose), oligosaccharides (i.e., amylose, amylopectin, glycogen).
Sugar derivatives that may be used in the methods and kits of this invention include, but are not limited to: alditols or aldose alcohol, which include erythritol, glucitol, sorbitol, or mannitol; ketoses, e.g., dihydroxyacetone, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, and tagatose; aminosugars such as glucosamine, galactosamine, N-acetylglucosamine, N-acetylgalactosamine, muramic acid, N-acetyl muramic acid, and N-acetyineuraminic acid (sialic acid); glycosides, such as glucopyranose and methyl-glucopyranose; and lactones, such as gluconolactone.
In certain embodiments of the invention, the non-ionic host cell buffer solution may include a mixture of sugars and derivatives thereof. One skilled in the art would be able to select suitable sugars to place in the mixture and to determine appropriate concentrations of these sugars and sugar derivatives to optimize the invention.
In certain embodiments of the methods and kits of this invention, at least one sugar or derivative thereof is added to host cells suspended in a non-ionic buffer solution prior to electrical treatment of the cells, wherein the concentration of the sugar or derivative thereof is in the range of about 0.1% to about 5%. Preferably, the concentration of the sugar or derivative thereof is about 2.0% to about 2.5%. In specific embodiments, the added sugar derivative is sorbitol and its concentration is about 2.5%.
As used herein, the term xe2x80x9celectrical treatmentxe2x80x9d includes any method of using electrical pulses or electrical discharges to introduce genes, fragments of genes, or other nucleic acids of interest into a cell. Electroporation methods are well known to those skilled in the art (See, e.g., Sambrook et al. 1987; Stratagene Instruction Manual for Epicurian Coli(copyright) Electroporation-Competent Cells 1997). Conditions for optimal efficiency can be determined by one skilled in the art.
In certain embodiments of the invention, bacterial cells are suspended in the non-ionic buffer solution comprising at least one sugar or sugar derivative prior to electrical treatment. Preferably, the bacterial cells are gram-negative bacterial cells (Davis, B. D et al., Microbiology: 3rd Edition (eds. Davis, B. D. et al. Harper and Row, 1995)). In a preferred embodiment of the invention, the gram-negative bacterial cells are E. coli. In a specific embodiment, the bacterial cell strain is XL1- Blue(trademark) (Stratagene Catalogue #200268).
As used herein, the term xe2x80x9cnucleic acids of interestxe2x80x9d includes, but is not limited to, nucleic acid sequences that encode functional or non-functional proteins, and fragments of those sequences, polynucleotides, or oligonucleotides. The nucleic acids of interest may be obtained naturally or synthetically, e.g., using PCR or mutagenesis. Further, the nucleic acids may be circular, linear, or supercoiled in their topology. Preferably, the nucleic acids are linear. Although not limited to such sizes, certain embodiments of this invention employ nucleic acids of interest ranging from about 3 kb to about 300 kb, depending on factors well known to those of skill in the art.
As used herein, the term xe2x80x9cpermitting the transfer of the nucleic acids of interest into the cellsxe2x80x9d may include transient transfer or permanent incorporation of the nucleic acids of interest into the bacterial cells by either autonomous replication or integration into the genome. One skilled in the art would be able to determine the optimal conditions to transfer the nucleic acids of interest, e.g., length of nucleic acids, pulse time. Typically, one skilled in the art may subject the host cells to electroporation for a certain period of time, thereby insuring the transfer of nucleic acids, but possibly sacrificing a large number of cells. This invention allows a larger number of host cells suspended in the above-described solutions to survive electroporation than cells suspended in the previously known solutions. Typically, subjecting E. coli to electrical transformation caused  greater than 90% of the cells to die; however, by practicing the methods of this invention, more of the cells will survive.
One skilled in the art would know how to select an appropriate media to promote growth of the transformed cells. The chosen media should propagate the transformed cells that either transiently express or have nucleic acids integrated into the host cells"" genome. Further, the media should be selected so as to assist the cells in recovering from the electrical treatment.
This invention also provides kits used in the practice of the methods of transferring nucleic acids into bacterial host cells according to this invention. In certain embodiments, the kits comprise transformation competent host cells suspended in a substantially non-ionic solution comprising at least one sugar or sugar derivative. In certain embodiments the kits, the concentration of the sugar or derivative thereof is in the range of about 0.1% to about 5%. In certain embodiments of the kits of this invention, the transformation competent host cells are bacterial cells, preferably E. coli. Other contents of the kit may include a control plasmid DNA for use in determining whether transformation has occurred.
The invention described above may be better understood by reference to the following examples. However, these examples are offered solely for the purpose of illustrating the invention, and should not be interpreted as limiting the invention.